Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative, facultatively anaerobic, motile bacillus that is responsible for a broad spectrum of illnesses observed in both humans and animals. While epidemiological surveys have demonstrated that B. pseudomallei is endemic to regions that typically border the equator, the incidence of disease is particularly high in South-East Asia and Northern Australia. In north eastern Thailand alone, an estimated 20% of community acquired septicemias and approximately 40% of deaths due to complications associated with bacterial sepsis can be attributed to this organism. The manifestations of melioidosis are commonly represented by acute, sub-acute and chronic illnesses, with the clinical manifestations often being mistaken for malaria, plaque, pneumonia and miliary tuberculosis. Infections are typically acquired via inhalation or aspiration, ingestion or via the direct contact of damaged surface tissues with contaminated waters or soils. Burkholderia mallei, the etiological agent of glanders, is a Gram-negative bacterium that is responsible for disease in donkeys, mules, horses and occasionally humans. Unlike the environmental saprophyte B. pseudomallei, however, B. mallei does not persist in nature outside of its soliped hosts. While B. mallei and B. pseudomallei are genotypically similar, significant phenotypic differences do exist between the two pathogenic species. Although glanders is one of the oldest diseases known to man, relatively little is known about the pathogenesis of disease caused by B. mallei. This phenomenon is primarily due to the lack of disease in North America along with the fact that B. mallei can be a particularly dangerous organism to study even in a controlled laboratory environment. Resistance to a variety of antimicrobial agents including penicillins, first- and second-generation cephalosporins and many of the aminoglycosides is characteristic of B. pseudomallei clinical isolates. With this in mind, the accurate identification of the organism, evaluation of the severity of the infection and antibiotic susceptibility testing are of paramount importance in devising successful chemotherapeutic protocols. While newer therapies that utilize combinations of ceftazidime-cotrimoxazole or amoxicillin-clavulanate for treatment are proving beneficial, the mortality rates associated with the acute septicemia and pulmonary forms of melioidosis are still unacceptably high. Typically, prolonged oral therapy is recommended to assure the resolution of infections while reducing the potential for recrudescence of disease. Characterization of the O-antigen of B. pseudomallei and B. mallei. Lipopolysaccharides (LPS), also commonly referred to as endotoxins, are a major component of Gram-negative cell envelopes. The ?barrier functions? provided by bacterial outer membranes are largely due to the presence of these molecules. LPS antigens expressed by smooth strains are composed of three covalently linked domains: an O-antigen, a core region and a lipid A moiety. O-antigens, consisting of oligosaccharide repeats, are the outermost domains of LPS molecules expressed on bacterial cell surfaces. Because of this, they are often a primary target of innate and acquired immune responses . Lipid A, the hydrophobic membrane-anchor component of LPS molecules, is the domain responsible for stimulating pathophysiological responses in mammals such as cytokine production, inflammation and shock. Host recognition of lipid A is mediated by the Toll-like receptor 4 (TLR4) complex. The complex, consisting of the TLR4 receptor and the MD2 and CD14 co-receptors is predominantly expressed by immune effectors such as monocytes and macrophages. TNF-a and IL-1a produced by LPS stimulated macrophages are two of the major pro-inflammatory cytokines responsible for the clinical manifestations of endotoxic shock. Lipopolysaccharides are important virulence determinants expressed by B. pseudomallei and B. mallei. Previous studies have demonstrated that the O-antigens expressed by these organisms are required for serum resistance. More importantly, the O-antigens have been also been identified as putative vaccine candidates for immunoprophylaxis against melioidosis and glanders. Studies have shown that the O-antigens expressed by B. mallei isolates are antigenically and structurally similar to those expressed by B. pseudomallei isolates. Structural analyses by Burtnick et al have demonstrated that like the predominant O-antigen expressed by B. pseudomallei isolates, the O-antigen expressed by B. mallei is an unbranched heteropolymer consisting of disaccharide repeats having the structure 3)-beta-D-glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which the 6-deoxy-a-L-talopyranose (L-6dTalp) residues are non-stoichiometrically modified by 2-O-acetyl and 2-O-methyl substitutions. Unlike B. pseudomallei, however, the L-6dTalp residues of the B. mallei O-antigen do not appear to be acetylated at the O-4 position. In contrast to many other Gram-negative pathogens, virulent isolates of B. mallei and B. pseudomallei appear to express a very limited repertoire of chemically distinct O-antigens. Based upon these findings, we have recently initiated studies to develop melioidosis and glanders vaccine candidates utilizing O-antigens purified from B. pseudomallei and B. mallei isolates. Burkholderia - macrophage interactions. The study of pathogen host cell interactions in vitro is an important tool to define and characterize virulence factors of intracellular bacterial pathogens. The major species of Burkholderia include B. pseudomallei; B. mallei and an avirulent environmentally stable isolate B. thailandensis. B. pseudomallei macrophage interactions have been extensively studied but there is little known about the interactions of B. mallei with macrophages. We have performed a comparative analysis of B. mallei and B. pseudomallei macrophage interactions using the murine macrophage cell line (RAW 264.7). Our findings show that although B. mallei is capable of invading and replicating in RAW cells it is less efficiently internalized and grows more slowly. The optimal multiplicity of infection is critical for permissive B. mallei intracellular growth. Moreover, B. mallei growth is significantly inhibited in activated macrophages. The differences between B. mallei and B. pseudomallei interactions with macrophages suggest species differences in virulence factors and indicate that more extensive comparative studies of both the pathogen and host could help to understand the molecular basis for these in Burkholderia species pathogenesis. We further tested differences in intracellular survival and multiplication among wild type and various mutants of B. mallei and B. pseudomallei. Eighteen mutants produced in each background of B. mallei and B. pseudomallei were tested in the RAW cell infection model. A type III secretion mutant of B. pseudomallei (strain 26bT3) showed marked differences in internalization and growth in RAW cells. An identical B. mallei type III secretion mutant (BMT3) and a B. mallei LPS mutant (GMrmlD) were incapable of growth in RAW cells. The results indicated that in vitro modeling of virulence using RAW macrophages is a simple and credible approach to screen Burkholderia mutants as a rational for analyses in animals and for more sophisticated molecular and biochemical characterization.